Detection of protein-DNA complex formation by time-resolved fluorescence depolarization of bound ethidium bromide.
We introduce the use of time-resolved fluorescence spectroscopy to probe the interaction between gene regulatory proteins and DNA. Changes in the decay kinetics of fluorescence polarization anisotropy of ethidium bromide bound to DNA segments report changes in hydrodynamic volume and shape which occurs upon complex formation between protein and DNA. We have used the decay of fluorescence polarization anisotropy as a spectroscopic handle on the interaction between several site-specific DNA-binding proteins involved in transcriptional regulation (the cro repressor of coliphage lambda, the lac repressor of Escherichia coli, and the RNA polymerase of coliphage T7) and their target DNA fragments ranging in length from 17 to 36 base pairs. The technique allows one to follow complex formation while varying solution conditions such as temperature, pH, ionic strength, and presence of effector molecules. Macromolecular concentrations ranging from 10(-7) to 10(-4) M can be used, allowing estimates of relative binding affinities. The magnitude of the observed rotational correlation times (phi obs) can be used to infer information about the size and shape of the complexes.
Cook
J
J
Department of Chemistry, University of Pennsylvania, Philadelphia 19104.
Holtom
G
G
Lu
P
P
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics; D004996:Ethidium; D005454:Fluorescence Polarization; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004996:Ethidium